In the field of gene recombination or the field of medicine, for introduction of a gene into cells of mammals including human, physical methods using electroporation or metal microparticles, chemical methods using nucleic acid, polycation, or liposome, and biological methods using virus-derived vectors for gene transfer (hereinafter, referred to as viral vectors) have been currently used. The viral vectors mean vectors obtained by altering natural viruses so that the viruses can transfer a desired gene or the like into a target, and the development of such vectors has been recently advanced. Viral vectors prepared by gene recombination technology are usually called recombinant viral vectors. Well-known examples of viruses which the recombinant viral vectors are derived from include viruses with envelopes, for example retrovirus, lentivirus, Sendai virus, and herpes virus, and viruses without envelopes (non-enveloped viruses), for example adenovirus, and adeno-associated virus (hereinafter, referred to as AAV).
In particular, AAV can infect a wide variety of cells including human cells, and AAV infects even non-dividing cells in which differentiation terminates, including blood cells, muscle cells, and nerve cells. In addition, since AAV is not pathogenic to human, it has a low risk of adverse effect. The virus particle of AAV is physicochemically stable. For these reasons, AAV has recently attracted attention to utility value as a vector for gene transfer used in gene therapy for the treatment of congenital genetic disease as well as the treatment of cancer or infection.
A method of producing a recombinant viral vector usually comprises introducing elements essential for formation of a virus particle in the form of a nucleic acid construct(s) into a cell to produce a cell having the ability to produce a virus (hereinafter, referred to as a virus-producing cell), and culturing the cell to express the elements essential for formation of the virus particle. In general, of the elements essential for formation of the virus particle, the elements that need to be provided in cis and the elements that can be provided in trans are separately introduced into a cell for viral production, thereby production of a wild-type virus and self-replication of a recombinant virus in a host infected with the virus are prevented (Patent Literature 1).
Hereinafter, as an example, a recombinant vector derived from AAV (hereinafter, referred to as rAAV) is specifically explained. An established method comprises 1) introduction of a rAAV plasmid in which an ITR placed at each end of the wild-type AAV genome is left and rep and cap genes are removed and 2) introduction of a plasmid for expression of rep and cap genes to provide Rep and Cap proteins in trans, and 3) infection with an adenovirus because AAV needs provision of supplemental elements for formation of the infectious virus particle from any virus called a helper virus, such as adenovirus, herpes virus, or vaccinia virus. A vector solution obtained by the above-mentioned method is theoretically contaminated with an adenovirus. In order to avoid the adenovirus contamination, a method of producing a vector comprising, instead of the above-mentioned 3), 3′) introduction of a helper plasmid expressing only elements essential for formation of an AAV virus particle among adenovirus-derived elements (Helper-free system) has been developed.
Virus-producing cells that have accomplished viral production are collected and homogenized to obtain a cell homogenate containing rAAV particles. The cell homogenate is subjected to a suitable step such as filtration with a filter, ultracentrifugation, chromatography, or ultrafiltration to purify the rAAV particles as a final product.
At present, as use of viral vectors is extended to the field of basic research or clinical application for gene therapy, a method of obtaining a virus particle with higher titer and higher purity is needed. Various improved methods are disclosed. For example, a method of enhancing production of virus particles and a release rate of the virus particles into a culture supernatant which comprises culturing a virus-producing cell under a stress condition in which a culture medium has an elevated pH is known (Patent Literature 1). Other methods comprise improvement of steps on and after purification of a produced virus. For purification of rAAV particles, a method of purifying rAAV particles of various serotypes without using ultracentrifugation or chromatography is proposed (Non-Patent Literature 1 and Non-Patent Literature 2). However, the said purification method comprises four steps: two-phase separation of a polyethylene glycol (PEG) phase and an aqueous phase, precipitation with PEG, treatment with chloroform, and dialysis; and thus it is complicated. In addition, a product purified by the purification method contains many bands on SDS-PAGE which are probably derived from impurities. Thus, the purification method is not adequate from the viewpoints of purity and simplicity. Therefore, there is still room for improvement in treatment of a virus-producing cell performed before purification of rAAV particles.